Project summary Limbal stem cells (LSCs) are required for reconstituting the corneal epithelium after injury. Injury to the cornea leading to substantial damage of LSCs or the LSC niche (LSCN) may lead to LSC deficiency (LSCD), a serious medical condition that causes corneal opacification, inflammation, vascularization, severe pain and conjunctivalization, and which can ultimately lead to complete loss of vision. The transplantation of LSCs expanded ex vivo onto the damaged cornea is a common surgical procedure that is carried out all around the world with an overall success rate of 60%- 70%. A major hurdle in culturing LSCs ex vivo is that they readily differentiate into central corneal epithelial cells, hampering their use for therapeutic applications and underscoring the need to regenerate the LSCN microenvironment in vitro for LSC expansion. Interestingly, our recently published work demonstrates that the LSCN is composed of an hyaluronan (HA) rich matrix which is required to support the stem cell phenotype. Aim 1 of this proposal will characterize the precise composition of the complex HA matrix present in the LSCN. Aim 2 will work towards reproducing the matrix present in the LSCN in culture conditions for ex vivo expansion of LSCs and also establishing how the HA matrix present in the LSCN regulates the LSC phenotype in vivo. Lymphatic vessels are present solely in the limbus (the HA rich region of the cornea) and the cornea is therefore avascular. Ocular injuries and inflammation, such as LSCD, often lead to irreversible corneal angiogenesis and lymphangiogenesis, which reduce corneal transparency, leading to vision loss and limiting the success of LSC transplantation. The lymphatic vessel marker Lyve-1 (LYmphatic Vessel Endothelial receptor 1) is a hyaladherin (a protein that specifically binds HA) and is expressed throughout the lymphatic system. Lyve-1 is capable of binding both soluble and immobilized HA; however, whether the HA/Lyve-1 interaction plays a role in lymphangiogenesis remains elusive. Interestingly, our preliminary data reveal that corneas of HA knock-out mice show only vestigial lymphatic vessels in the limbus, suggesting that an HA scaffold may be necessary for corneal lymphangiogenesis. HA up-regulation throughout the cornea precedes exacerbated extension of lymphatic vessels, and injecting HA into the corneal stroma leads to the extension of lymphatic vessels. Therefore, we postulate that HA plays an important role in regulating corneal lymphangiogenesis. Aim 3 proposes to identify the mechanism by which HA within the corneal limbus regulates the development of lymphatic vessels.